PROJECT SUMMARY Esophageal adenocarcinoma (EAC) is one of the deadliest malignancies, and its incidence has strikingly increased 6-8 fold in Western countries (including the United States, UK and several European countries) over the past 4 decades. Despite new insights gained from recent genomic analyses, meaningful therapeutic improvements have not occurred and the 5-year survival of EAC has remained extremely low (~20%). Therefore, alternative research approaches, including advanced epigenomic studies, are desperately needed to understand the molecular basis of EAC for developing novel treatment regimens. Barrett?s esophagus (BE) is a premalignant condition and is considered as the obligate precursor lesion of EAC. During Barrett?s esophagus-associated neoplastic evolution, benign BE first becomes dysplastic and then progresses to EAC. Therefore, BE serves as an ideal pre-malignant model for the investigation of the step-wise neoplastic evolution of esophageal epithelial cells. However, our understanding of the molecular mechanisms promoting BEAN remains limited, with key questions (e.g., the primary drivers for the malignant transformation of BE into EAC) still unaddressed. We and others have shown that malignant transformation is accompanied by genome-wide gains and losses of enhancers and super-enhancers, which are occupied and regulated by upstream master regulator transcription factors (MRTFs). Indeed, our recent studies demonstrated profound alterations in both enhancer usage and MRTF activity between normal gastroesophageal junction (NGEJ), BE and EAC samples. Particularly, we have identified a set of EAC-specific MRTFs (ELF3, KLF5, GATA6, EHF). Pilot experiments have shown that these 4 MRTFs co-occupy hundreds of EAC-specific enhancers and super-enhancers, indicating they may regulate the EAC transcriptome. Moreover, these EAC-specific MRTFs are highly and uniquely expressed in EAC compared with normal GEJ or BE samples and are functionally required for EAC cell proliferation. Based on these findings, we hypothesize that EAC-specific MRTFs directly promote the malignant transformation of BE cells by rewiring enhancers and super-enhancers across the epigenome, activating signaling pathways and cellular processes essential for EAC development. We will test this hypothesis by investigating the biological functions of MRTFs in human BE-derived 3D organoids. In addition, we will study the mechanistic basis of the strong association between obesity and EAC by focusing on the regulatory loop of MRTF and fatty-acid synthesis, which is the key downstream pathway identified by our preliminary data. These investigations promise to establish primary driving forces of BE-associated neoplasia evolution and uncover epigenomic mechanisms underlying esophagus transformation, which will fundamentally transform our insights into the biology of esophageal cancer. More importantly, successful execution of this proposal may identify potential avenue for the prevention and early intervention of EAC by targeting fatty-acid synthesis pathway in the high-risk individuals (e.g., refractory and/or high-grade BE patients) with obese condition.